Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

大多数遗传疾病源于隐性点突变，需要纠正而不是破坏致病等位基因才能使患者受益。碱基编辑有可能直接修复点突变并提供基因功能的治疗性恢复。跨膜通道样 1 基因 ( TMC1 ) 的突变可导致显性或隐性耳聋。我们开发了一种碱基编辑策略来治疗 Baringo 小鼠，这些小鼠在 Tmc1 中携带隐性的、功能丧失的点突变（c.A545G；导致 p.Y182C 的替代），导致耳聋。tmc1编码一种在内耳感觉毛细胞中形成机械敏感离子通道的蛋白质，是正常听觉功能所必需的。我们发现 Baringo 小鼠的感觉毛细胞完全丧失了听觉感觉转导。为了修复突变，我们在 Baringo 小鼠胚胎成纤维细胞中测试了几种优化的胞嘧啶碱基编辑器（CBEmax 变体）和引导 RNA。我们使用分裂内含肽递送系统将最有前途的 CBE，源自激活诱导的胞苷脱氨酶 (AID)，打包到双腺相关病毒 (AAV) 中。双AID-CBEmax的AAV注射入小鼠巴拉戈的内耳在出生后第1注射的小鼠表现出高达51％的返原TMC1 c.A545G点突变野生型序列（c.A545A中）Tmc1转录本。的修复TMC1体内恢复内毛细胞的感官转导和毛细胞的形态和瞬时救出低频注射后听力4周。这些发现为隐性听力损失的潜在一次性治疗奠定了基础，并支持进一步发展碱基编辑以纠正致病性点突变。